The present invention relates to thermostable DNA polymerases, polynucleotide sequences encoding them, and methods for their manufacture.
DNA polymerases are well known, and are generally commercially available in purified form. These enzymes are useful in a wide range of laboratory processes, especially in molecular biology. Primer extension techniques, nucleic acid sequencing and the polymerase chain reaction (PCR) all employ such enzymes.
Thermostable DNA polymerases are particularly useful in a number of these techniques, as thermostable enzymes are able to be used at relatively high temperatures. This has benefits with respect to fidelity of primer binding, for example, owing to the high stringency of the conditions employed. Of known enzymes, the DNA polymerase isolated from Thermus aquaticus (Taq) is perhaps the best characterized. In addition, DNA polymerases have been isolated from other thermophilic bacteria, such as Thermococcus litoralis, Thermus thermophilus (T.th) and Pyrococcus furiosus (Pfu).
The available, purified enzymes have a defined range of different properties and limitations. Taq DNA polymerase, for example, has no proof-reading activity, which results in errors during DNA amplification. Native Pfu DNA polymerase has lower thermostability than Taq DNA polymerase, so that it loses activity faster than Taq DNA polymerase under similar conditions. In addition, Pfu DNA polymerase has a proofreading ability which reduces the rate of incorporation of the enzyme, which in turn reduces the processivity of the enzyme. The reduced thermostability in combination with the reduced processivity has the effect of reducing the length of the fragments that may be amplified by Pfu DNA polymerase, in comparison with Taq DNA polymerase. Therefore, neither Taq nor Pfu DNA polymerase have optimum properties for use in DNA amplification procedures.
Combinations of enzymes, such as mixtures of Taq DNA polymerase and Pfu DNA polymerase, have been used to try to obtain the combined benefits of both enzymes, but such mixtures are still subject to the limitations of each individual enzyme in the mixture. Moreover, commercial products using two enzymes are inevitably more expensive and there is a more stringent burden upon quality control to ensure that both enzymes have the necessary level of enzyme activity.
Thus, there is still a need for a single thermostable DNA polymerase with a combination of desirable properties that do not occur in nature.
We have now, surprisingly discovered that a functional, thermostable, chimaeric enzyme that overcomes the above problems may be obtained by combining functionalities of different enzymes.
Thus, in a first aspect, the present invention provides an enzyme which has the properties of thermostability, DNA polymerase activity and proof-reading, said properties being derived from at least two different sources, wherein the properties are preferably in synergistic combination.
In an alternative aspect, the present invention provides an enzyme which has the properties of thermostability, DNA polymerase activity and proof-reading, said properties being derived from at least two different polypeptides.